Abstract:

One of the greatest challenges of implementing fast molecular detection methods
as part of Legionella surveillance systems is to limit detection to live cells.
In this work, a protocol for sample treatment with propidium monoazide (PMA) in
combination with quantitative PCR (qPCR) has been optimized and validated for L.
pneumophila as an alternative of the currently used time-consuming culture
method. Results from PMA-qPCR were compared with culture isolation and
traditional qPCR. Under the conditions used, sample treatment with 50 μM PMA
followed by 5 min of light exposure were assumed optimal resulting in an average
reduction of 4.45 log units of the qPCR signal from heat-killed cells. When
applied to environmental samples (including water from cooling water towers,
hospitals, spas, hot water systems in hotels, and tap water), different degrees
of correlations between the three methods were obtained which might be explained
by different matrix properties, but also varying degrees of non-culturable
cells. It was furthermore shown that PMA displayed substantially lower
cytotoxicity with Legionella than the alternative dye ethidium monoazide (EMA)
when exposing live cells to the dye followed by plate counting. This result
confirmed the findings with other species that PMA is less membrane-permeant and
more selective for the intact cells. In conclusion, PMA-qPCR is a promising
technique for limiting detection to intact cells and makes Legionella
surveillance data substantially more relevant in comparison with qPCR alone. For
future research it would be desirable to increase the method's capacity to
exclude signals from dead cells in difficult matrices or samples containing high
numbers of dead cells